Bacterial cells, such as E. coli, are commonly used for producing recombinant proteins. There are many advantages to using bacterial cells, such as E. coli, for producing recombinant proteins particularly due to the versatile nature of bacterial cells as host cells allowing the gene insertion via plasmids. E. coli have been used to produce many recombinant proteins including human insulin.
Despite the many advantages to using bacterial cells to produce recombinant proteins, there are still significant limitations including the tendency of bacterial cells to lyse during expression of a recombinant protein of interest. This lysis phenotype may be seen in wild-type bacterial cells and also genetically engineered cells, such as cells which are deficient in bacterial proteases. Proteases play an important role in turning over old, damaged or misfolded proteins in the E. coli periplasm and cytoplasm. Bacterial proteases act to degrade the recombinant protein of interest, thereby often significantly reducing the yield of active protein. Therefore, the reduction of protease activity is desirable to reduce proteolysis of proteins of interest. However, bacterial strains lacking proteases, such as Tsp (also known as Prc), also exhibit cell lysis.
Tsp (also known as Prc) is a 60 kDa periplasmic protease. The reduction of Tsp (prc) activity is desirable to reduce the proteolysis of proteins of interest. However, it was found that cells lacking the protease prc show thermosensitive growth at low osmolarity. Hara et al isolated Tsp deficient strains which were thermoresistant revertants containing extragenic suppressor (spr) mutations (Hara et al., Microbial Drug Resistance, 2: 63-72 (1996)). Spr is an 18 kDa membrane bound periplasmic protease and the substrates of spr are Tsp and peptidoglycans in the outer membrane involved in cell wall hydrolysis during cell division. The spr gene is designated as UniProtKB/Swiss-Prot P0AFV4 (SPR_ECOLI). Protease deficient bacterial strains carrying a mutant spr gene have been described in Chen et al (Chen C, Snedecor B, Nishihara J C, Joly J C, McFarland N, Andersen D C, Battersby J E, Champion K M. Biotechnol Bioeng. 2004 Mar. 5; 85(5):463-74) which describes the construction of E. coli strains carrying different combinations of mutations in prc (Tsp) and another protease, DegP, created by amplifying the upstream and downstream regions of the gene and ligating these together on a vector comprising selection markers and a sprW174R mutation.
It has been surprisingly found that a gram-negative bacterial cell carrying a mutant spr gene and a wild-type Tsp gene provides a cell having reduced lysis. Accordingly, the present inventors have provided a new strain having advantageous properties for producing a protein of interest.
It was surprising that cells according to the present invention show advantageous growth and protein yield phenotype because spr and Tsp are known to be mutual suppressors and, therefore, it would be predicted that if one is allowed to dominate the cell may exhibit a poor growth phenotype, such as becoming leaky or show increased propensity to cell lysis. However, the cells of the present invention exhibited a significant reduction in cell lysis phenotype compared to wild-type cells and cells comprising a knockout mutated Tsp gene.